Method for the treatment of hypotension induced by a cytokine or bacterial endotoxin using s-alkyl- isothioureido-amino acids

ABSTRACT

Inhibitors of nitric oxide formation from arginine useful for treating hypotension, inflammation, stroke and to restore vascular contractile sensitivity to the effects of α 1  -adrenergic agonists are physiologically active compounds including N.sup.δ -substituted ornithine or N.sup.ε -substituted lysine moieties or monoalkyl carbon-substituted N.sup.δ -substituted ornithine or N.sup.ε -substituted lysine moieties, having the formula ##STR1## wherein R is (CH 2 ) y  CH 3  or H, R&#39; is CH 2  or C(H)(CH 2 ) y  CH 3 , and R&#34; is CH 2  or C(H)(CH 2 ) y  CH 3 , with y ranging from 0 to 5, and x is 0 or 1 and wherein none or only one of R, R&#39; and R&#34; provides an alkyl substituent on ornithine or lysine moiety, and wherein Q is alkyl having 1 to 5 carbon atoms, and physiologically acceptable acid addition salts thereof.

This is a continuation of application Ser. No. 08/152,010 filed on Nov.11, 1993, now U.S. Pat. No. 5,364,881.

TECHNICAL FIELD

The invention is directed to novel inhibitors of biological nitric oxideformation.

BACKGROUND OF THE INVENTION

For several decades nitroglycerin has been administered to humans as avasodilating agent in the treatment of cardiovascular disease. It hasbeen shown that nitroglycerin so administered is converted in the bodyto nitric oxide which is the pharmacologically active metabolite.Recently, nitric oxide has been shown to be formed enzymatically as anormal metabolite from arginine in vascular endothelium to provide animportant component of endothelium-derived relaxing factors (EDRFs)which are currently being intensively studied as participating inregulation of blood flow and vascular resistance. Macrophages have alsobeen shown to produce nitric oxide in the body as a component of theircell killing and/or cytostatic function.

More recently it has been established that the enzyme forming nitricoxide from arginine, i.e., nitric oxide synthase, occurs in at least twodistinct forms, namely constitutive forms and an inducible form. Theconstitutive forms are present in normal endothelial cells, neurons andsome other tissues. Formation of nitric oxide by constitutive forms inendothelial cells is thought to play a role in normal blood pressureregulation. The inducible form of nitric oxide synthase has been foundto be present in activated macrophages and is induced in endothelialcells and vascular smooth muscle cells, for example, by variouscytokines and/or microbial products. It is thought that in sepsis orcytokine-induced shock, overproduction of nitric oxide by the inducibleform of nitric oxide synthase plays an important role in the observedlife-threatening hypotension. Furthermore, it is thought thatoverproduction of nitric oxide by the inducible form of nitric oxidesynthase is a basis for insensitivity to clinically used pressor agentssuch as α₁ -adrenergic agonists in the treatment of septic orcytokine-induced shock patients. Moreover, it is thought thatoverproduction of nitric oxide by inducible form of nitric oxidesynthase is involved in inflammation incident to an immune response.

SUMMARY OF THE INVENTION

It is an object of one embodiment of the invention herein to providenovel arginine or citrulline antagonists which inhibit constitutive formof nitric oxide synthase or inducible form of nitric oxide synthase orboth.

The novel compounds herein are physiologically active compoundsincluding N.sup.δ -substituted ornithine or N.sup.ε -substituted lysinemoieties or monoalkyl carbon-substituted N.sup.δ -substituted ornithineor N.sup.ε -substituted lysine moieties, having the formula ##STR2##wherein R is (CH₂)_(y) CH₃ or H, R' is CH₂ or C(H)(CH₂)_(y) CH₃, and R"is CH₂ or C(H)(CH₂)_(y) CH₃, with y ranging from 0 to 5, and x is 0 or 1and wherein none or only one of R, R' and R" provides an alkylsubstituent on ornithine or lysine moiety, and wherein Q is alkyl having1 to 5 carbon atoms, and physiologically acceptable acid addition saltsthereof.

Preferably Q is methyl.

Preferred compounds are S-methyl-L-thiocitrulline (the above structuralformula where R is H, R' is CH₂, R" is CH₂, x is O, and Q is CH₃) andS-methyl-L-homothiocitrulline (the above structural formula where R isH, R' is CH₂, R" is CH₂, x is 1 and Q is CH₃) and the physiologicallyacceptable acid addition salts thereof.

The physiologically acceptable acid addition salts include, for example,acetate, hydrochloride, sulfate, phosphate, succinate, citrate andpropionate acid addition salts.

The term "physiologically active" refers to L-enantiomer whether pure orin admixture with D-enantiomer. The D-enantiomers are notphysiologically active or are much less active than the L-enantiomers.Thus, in the D,L-compounds only the L-enantiomer portion isphysiologically active.

Preferably the compounds are pharmaceutically pure, i.e., more than 99%by weight pure (on a water free basis) and contain from 99% to 100% byweight of L-enantiomer (on an L- and D-enantiomer basis).

It is an object of another embodiment herein to provide methods forprophylaxis or treatment of a subject for systemic hypotension orexpected systemic hypotension caused by pathological overproduction ofnitric oxide from arginine by nitric oxide synthase induced in vascularcells in said subject by a cytokine or by a bacterial endotoxin. One ofthese methods comprises administering to said subject a therapeuticallyeffective amount of a compound of the invention herein. Another of thesemethods comprises administering to said subject a conventional amount ofat least one α₁ -adrenergic agonist and an amount of compound of theinvention herein effective to restore vascular contractile sensitivityto the effects of said α₁ -adrenergic agonist.

It is an object of another embodiment herein to suppress an immuneresponse in a subject in need of said suppressing, e.g., where theimmune response is part of a pathological inflammatory response. Thismethod comprises administering to a subject in need of said suppressingof an immunosuppressive effective amount, e.g., an inflammationameliorating amount, of compound of the invention herein.

It is an object of still another embodiment herein to provide a methodof prophylaxis or treatment of a subject for a stroke. This methodcomprises administering to said subject of a therapeutically effectiveamount, e.g., a neuronal cell protecting amount, of a compound of theinvention herein.

The term "subject" is used herein to mean any mammal, including humans,where nitric oxide formation from arginine occurs.

The term "prophylaxis" is used herein to mean to prevent or delay theoccurrence of a condition or to ameliorate the symptoms of a conditionshould it occur compared to where prophylaxis is not carried out.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of product formation versus time, depicting results ofExample III.

FIG. 2 is a graph of product formation versus time, depicting results ofExample IV.

FIG. 3 is a graph of systolic blood pressure (denoted BP) versus time,depicting results of Example V.

FIG. 4 is a graph of product formation versus time, depicting results ofExample VIII.

FIG. 5 is a graph of product formation versus time, depicting results ofExample IX.

DETAILED DESCRIPTION

We turn now in more detail to the novel compounds herein.

As indicated above, one group of compounds herein consists of thosewhere none of R, R' and R" provides an alkyl substituent on ornithine orlysine moiety. Where this is the case, the compounds herein are thoseincluding ornithine or lysine moieties without monoalkylcarbon-substitution thereon. Ornithine moiety is the case where x equalszero. Lysine moiety is the case where x is 1.

As indicated above, another group of compounds herein consists of thosewhere one of R, R' and R" provides an alkyl substituent on ornithine orlysine moiety. Where this is the case, the compounds herein are thoseincluding monoalkyl carbon-substituted ornithine or lysine moieties.Monoalkyl carbon-substituted ornithine moiety is the case where x equalszero. Monoalkyl carbon-substituted lysine moiety is the case where x is1.

Examples of specific compounds having the structural formula set forthabove where none of R, R' and R" provides an alkyl substituent onornithine or lysine moiety are S-methyl-L-thiocitrulline,S-ethyl-L-thiocitrulline, S-propyl-L-thiocitrulline,S-isopropyl-L-thiocitrulline, S-cyclopropyl-L-thiocitrulline,S-butyl-L-thiocitrulline, S-isobutyl-L-thiocitrulline,S-methylcyclopropyl-L-thiocitrulline, S-pentyl-L-thiocitrulline,S-isopentyl-L-thiocitrulline, S-methyl-L-homothiocitrulline,S-ethyl-L-homothiocitrulline, S-propyl-L-homothiocitrulline,S-isopropyl-L-homothiocitrulline, S-isobutyl-L-homothiocitrulline,S-methylcyclopropyl-L-homothiocitrulline, S-pentyl-L-homothiocitrullineand S-isopentyl-L-homothiocitrulline.

S-Methyl-L-thiocitrulline may be prepared as set forth in Example I.S-Methyl-L-homothiocitrulline may be prepared as set forth in ExampleII. Other S-alkyl-L-thiocitrullines may be prepared as set forth inExample I by substituting the corresponding iodoalkane for theiodomethane used in Example I (i.e., iodoethane to produceS-ethyl-L-thiocitrulline, iodopropane to produceS-propyl-L-thiocitrulline, etc.) Other S-alkyl-L-homothiocitrullines maybe prepared as set forth in Example II by substituting the correspondingiodoalkane for the iodomethane used in Example II.

Examples of specific compounds where one of R,R' and R" provides analkyl substituent on ornithine or lysine moiety areα-methyl-S-methyl-L-thiocitrulline (the above structural formula where Ris CH₃, R' is CH₂ and R" is CH₂ and x is zero and Q is CH₃),α-propyl-S-propyl-L-thiocitrulline, β-methyl-S-methyl-L-thiocitrulline(the above structural formula where R is H, R' is C(H)(CH₃) and R" isCH₂ and x is zero and Q is CH₃), β-ethyl-S-isobutyl-L-thiocitrulline,γ-methyl-S-methyl-L-thiocitrulline (the above structural formula where Ris H, R' is CH₂ and R" is C(H)(CH₃) and x is zero and Q is CH₃),γ-butyl-S-methyl-L-thiocitrulline,α-methyl-S-methyl-L-homothiocitrulline (the above structural formulawhere R is CH₃, R' is CH₂ and R" is CH₂ and x is 1 and Q is CH₃),α-pentyl-S-methyl-L-homothiocitrulline,β-methyl-S-methyl-L-homothiocitrulline (the above structural formulawhere R is H, R' is C(H)(CH₃) and R" is CH₂ and x is 1 and Q is CH₃), β-methyl-S-pentyl-L-homothiocitrulline,Y-methyl-S-methyl-L-homothiocitrulline andγ-ethyl-S-ethyl-L-homothiocitrulline (the above structural formula whereR is H, R' is CH₂ and R" is C(H)(CH₂ CH₃) and x is 1 and Q is C₂ H₅) andthe corresponding D,L-compounds.

The N.sup.δ -benzyloxycarbonyl-L-ornithine of Example I may be preparedby reacting benzyloxycarbonyl chloride with copper salt of L-ornithine.The N.sup.ε -benzyloxycarbonyl-L-lysine of Example II may be prepared byreacting benzyloxycarbonyl chloride with copper salt of L-lysine. Theseare well known reactions.

The D,L-compounds where one of R, R' and R" provides an alkylsubstituent on ornithine or lysine moiety are prepared by utilizing theappropriate one of α-alkyl-DL-ornithine, β-alkyl-DL-ornithine,γ-alkyl-DL-ornithine, α-alkyl-DL-lysine, β-alkyl-DL-lysine orγ-alkyl-DL-lysine in place of L-ornithine or L-lysine in the synthesisof the compounds where none of R, R' and R" provides an alkylsubstituent on ornithine or lysine moiety. The synthesis of theα-alkylornithines, the β-alkylornithines, the γ-alkylornithines, theα-alkyllysines, the β-alkyllysines and the γ-alkyllysines are describedin Griffith U.S. patent application Ser. No. 07/889,345.α-Methyl-DL-ornithine is commercially available from Sigma Chemicals,St. Louis, Mo. The synthesis of RS-β-methyl-DL-ornithine andRS-γ-methyl-DL-ornithine are described in detail in said Ser. No.07/889,345. L-compounds can be made by using L-reactants which can beobtained by resolution of the corresponding D,L-compounds. Admixtures of50 to 100% L-enantiomer with the remainder being D-enantiomer can beprepared by admixing L-compound with D,L-compound. Admixtures containingless than 50% L-enantiomer can be made by synthesizing D-enantiomer (bystarting with D-compound reactant) and admixing this with L-compound orD,L-compound.

The compounds herein are not biologically active as arginine analogswith respect to protein synthesis. Therefore, there is no possibility ofthese interfering with incorporation of arginine into proteins.

We turn now to the methods herein.

As previously indicated, embodiments herein are directed to methods oftreatment of a subject for systemic hypotension caused by pathologicaloverproduction of nitric oxide from arginine by the enzyme nitric oxidesynthase induced in vascular cells and, perhaps, other tissues, in saidsubject with a cytokine or by a bacterial endotoxin. The inducement bycytokines, e.g., gamma-interferon, tumor necrosis factor, interleukin-1or interleukin-2 can occur because of therapy with said cytokines, e.g.,chemotherapeutic treatment with tumor necrosis factor or interleukin-2.In such therapy, the cytokine is administered in conventional amountsfor said therapy. However, whereas the period for administration of saidtherapy is normally limited by the eventual occurrence of severehypotension and vascular leak, the method herein allows concomitantadministration of compound of the invention herein (the term prophylaxisincludes said concurrent administration) to delay or eliminate theoccurrence of these symptoms. The inducement by endotoxin from bacterialinfection or other bacterial toxin is known as septic shock and is theleading cause of death in intensive care units, some 250,000 deaths inone year recently in the U.S. This can be an expected condition in caseswhere the immune system is compromised, e.g., because ofimmunosuppression therapy or in AIDS. Septic shock also occurs inimmunocompetent people.

We turn now to the method herein for prophylaxis or treatment of asubject for systemic hypotension caused by biological overproduction ofnitric oxide from arginine by nitric oxide synthase induced in vascularcells in said subject with a cytokine or by a bacterial endotoxinwherein the method comprises administering to said subject atherapeutically effective amount of a compound of the invention herein.For treatment of systemic hypotension which is already occurring, thecompound is administered in a blood pressure raising amount, generally 1mg/kg to 100 mg/kg for L-enantiomer (preferably 2 mg/kg to 20 mg/kg forS-methyl-L-thiocitrulline and 2 mg/kg to 20 mg/kg forS-methyl-L-homothiocitrulline) by a route of administration obtaining afast response, normally parenteral, preferably intravenous. Fortreatment in cases where systemic hypotension is expected (i.e., forprophylaxis, i.e., prevention or delay of the condition occurring or toprovide ameliorated occurrence of the condition), administration is toprovide a plasma level of compound of invention herein sufficient toeliminate or delay the occurring of the hypotension or to reduce theseverity of the hypotension which occurs, generally a plasmaconcentration ranging from 1 μM to 100 μM for L-enantiomer (preferably10 μM to 50 μM for S-methyl-L-thiocitrulline and 10 μM to 50 μM forS-methyl-L-homothiocitrulline) by a route of administration which can beparenteral (e.g., intravenous) but also can be oral (doses to providethis concentration may be determined by considering the half-life of thecompounds in the body).

We turn now to the method herein for treatment of a subject for systemichypotension caused by pathological overproduction of nitric oxide fromarginine by nitric oxide synthase induced in vascular cells in saidsubject with a cytokine or by a bacterial endotoxin wherein the methodcomprises administering to a subject in need of said treatment of aconventional amount of at least one α₁ -adrenergic agonist and an amountof compound of the invention herein effective to restore vascularcontractile sensitivity to the effects of said α₁ -adrenergic agonists.The α₁ -adrenergic agonists are used for the same purpose now (i.e., toincrease blood pressure in a hypotensive patient) but eventually stopworking because of loss of vascular contractile sensitivity. The α₁-adrenergic agonists are used in the same dosages as they are used nowfor the same purpose, i.e., in conventional therapeutically effectiveamounts. Suitable α₁ -adrenergic agonists are epinephrine,norepinephrine, dopamine, phenylephrine, metaraminol, methoxamine,ephedrine, and mephentermine. Use with the pressor angiotensin II isalso envisioned. Doses for dopamine typically range from 2 μg/kg/min to50 μg/kg/min. Doses for epinephrine typically range from 0.25 mg to 1.0mg. Doses for norepinephrine typically range from 2 μg/min to 4 μg/minand are typically used if dopamine dose exceeds 20 μg/kg/min. Doses forphenylephrine can range from 0.1 to 10 μg/kg. Doses for angiotensin IIcan range from 0.01 to 1 μg/kg. The route of administration of the mostpopular α₁ -adrenergic agonists (epinephrine, norepinephrine anddopamine) is intravenous and for the others the route of administrationis intravenous or in some cases subcutaneous. The compound of theinvention herein is administered in an amount effective to restorevascular contractile sensitivity to the effects of the α₁ -adrenergicagonist (i.e., to increase and/or prolong the efficacy of the α₁-adrenergic agonists), generally 1 mg/kg to 100 mg/kg for L-enantiomer(preferably 2 mg/kg to 20 mg/kg for S-methyl-L-thiocitrulline and 2mg/kg to 20 mg/kg for S-methyl-L-homothiocitrulline) by a route ofadministration obtaining a fast response, normally parenteral,preferably intravenous.

We turn now to the method herein for suppressing an immune response,e.g., where the immune response is part of an inflammatory response, ina subject in need of said suppressing, said method comprisingadministering to a subject in need of said suppressing animmunosuppressive effective amount of compound of the invention herein.This method may be directed to prophylaxis or treatment of a subject forinflammation, e.g., arising from autoimmune conditions includingrheumatoid arthritis and from host-defense immune mechanisms, e.g.,allograft rejection reactions, caused by immunologically induced nitricoxide production in immune cells, said method involving inhibiting saidnitric oxide production in said cells by administering to a subjectpossibly developing or having such inflammation, a nitric oxidesynthesis inhibiting therapeutically effective (inflammationattenuation) amount of compound of the invention herein. The dosages ofL-enantiomer compound herein for use in this method generally range from1 mg/kg to 1000 mg/kg (preferably 2 mg/kg to 200 mg/kg forS-methyl-L-thiocitrulline and 2 mg/kg to 200 mg/kg forS-methyl-L-homothiocitrulline). Methods of administration include oral,intramuscular, subcutaneous and intravenous. The dosages set forth aboveare daily dosages and are administered for a period of time to causesuppression of immune response and attenuation of inflammation, i.e.,two days or more, e.g., for two days to three weeks.

We turn now to the method herein for prophylaxis or treatment of asubject for a stroke wherein the method comprises administering to saidsubject of a therapeutically effective amount of compound of theinvention herein. For a stroke in progress, administration is preferablywithin 6 hours of the onset of the stroke, very preferably within 4hours of the onset of the stroke. Since time is of the essence,administration typically is as soon as practical after diagnosis. Thetherapeutically effective amount is a neuronal cell protecting amount,i.e., an amount which causes increase in neuronal cell survival comparedto where the stroke is untreated. Generally, the dose for L-enantiomerranges from 1 mg/kg to 100 mg/kg (preferably 2 mg/kg to 20 mg/kg forS-methyl-L-thiocitrulline and 2 mg/kg to 20 mg/kg forS-methyl-L-homothiocitrulline). Administration is by a route offering afast response, e.g., parenteral, preferably intravenous orintraarterial. Prophylaxis involves treatment of those of high risk fora stroke because of medical history and administration is that amountsufficient to provide an uninterrupted plasma level of compound hereinin a neuronal cell protecting concentration, generally 1 μM to 100 μMfor L-enantiomer (preferably 10 μM to 50 μM forS-methyl-L-thiocitrulline and 10 μM to 50 μM forS-methyl-L-homothiocitrulline) and administration is preferably carriedout orally on a daily basis.

Dosages are given above for the L-enantiomer. For admixtures ofL-enantiomer and D-enantiomer, dosages are calculated by dividing thosegiven above by the weight percent of L-enantiomer in the admixture.

The invention is illustrated in the following examples.

EXAMPLE 1 Synthesis of S-Methyl-L-thiocitrulline

N.sup.δ -(Benzyloxycarbonyl)-L-ornithine tert-butyl ester was preparedaccording to a general published procedure (Bodanszky, M.; Bodanszky,A.; The Practice of Peptide Synthesis; Spring Verlag: New York, 1984).Specifically, N.sup.δ -(benzyloxycarbonyl)-L-ornithine (10.0 gm, 37.6mmol, Sigma Chemicals, Inc.) was mixed with tert-butyl acetate (564 ml)and perchloric acid (5.91 ml, 69-72% aqueous solution) at roomtemperature. The reaction mixture became homogenous after 15 minutes andwas stirred at room temperature for 2 days. Water (300 ml) was added tothe reaction mixture, and the two layers obtained were separated. Theorganic layer was extracted twice (2×200 ml) with water, and thecombined aqueous layers were adjusted to pH 9.5-10.0 with 50% NaOH. Theaqueous layer was then filtered to remove a small amount of insolublematerial, and the filtrate was extracted with ethyl acetate (3×300 ml).The combined ethyl acetate extracts were dried over MgSO₄ and wereconcentrated by rotary evaporation at reduced pressure to yield 7.5 g ofN.sup.δ -(benzyloxycarbonyl)-L-ornithine tert-butyl ester as an oil (65%yield). ¹ H NMR(DCCl₃) δ 1.44 (s,9H), 1.5-1.9 (m,4H), 3.23 (t,2H), 3.31(t,1H) , 5.09 (s,2H and s,1H broad), 7.35 (s,5H).

N.sup.α -(tert-Butyloxycarbonyl)-N.sup.δ-(benzyloxycarbonyl)-L-ornithine tert-butyl ester

N.sup.δ -(Benzyloxycarbonyl-L-ornithine tert-butyl ester (7.50 g, 23.3mmol) was dissolved in 27 ml of methylene chloride, and the solution wascooled to 0° C. To that solution was added dropwise tert-butylpyrocarbonate (5.87 g, 26.9 mmol) in 10 ml of methylene chloride. Thereaction mixture was stirred at 0° C. for 1 hour, and stirring wascontinued for 3 additional hours at room temperature. The solvent wasevaporated at reduced pressure and the oily residue was chromatographedon a column of silica gel (25 cm×30 mm) using hexane:ethyl acetate (3:1)as solvent. Fractions of approximately 5 ml were collected, and thosecontaining product were identified by thin layer chromatography (seebelow). Chromatographic fractions containing product were pooled andevaporated to a thick oil by rotary evaporation at reduced pressure. Theyield was 8.63 gm (93.5%). ¹ H NMR (DCCl₃) δ 1.44 (s,9H), 1.46 (s,9H),1.5-1.9 (m,4H), 3.23 (t,2H), 4.17 (t, 1H), 4.88 (s,1H broad), 5.09 (s,2Hand s,1H broad) 7.35 (s,5H).

Thin layer chromatography was carried out on silica gel plates usinghexane:ethyl acetate (3:1) as solvent. N.sup.δ-(benzyloxycarbonyl)-N.sup.α -(tert-butyloxycarbonyl)-L-ornithinetert-butyl ester chromatographed with an R_(f) =0.33 and was detected byfluorescence using a hand-held UV light.

N.sup.δ -(tert-butyloxycarbonyl-L-ornithine tert-butyl ester

The ester from the previous step (8.63 gm, 20.45 mmol) was dissolved in50 ml of methanol and 10% Pd/C catalyst (0.90 gm) was added. The mixturewas hydrogenated on a Parr hydrogenator at 20 psi H₂ for 4 hr. Followingreduction, the reaction mixture was filtered over Celite and evaporatedto dryness by rotary evaporation at low pressure to provide N.sup.α-(tert-butyloxycarbonyl)-L-ornithine tert-butyl ester as an oil. Theyield was 5.88 gm (100%). ¹ H NMR (DCCl₁₃) δ 1.44 (s,9H), 1.46 (s,9H),1.5-1.9 (m,4H), 2.73 (t,2H), 4.17 (t,1H), 5.20 (s,1H broad).

N.sup.α -(tert-butyloxycarbonyl)-N.sup.δ -(thioureido)-L-norvalinetert-butyl ester

This product was prepared as described by Feldman (Feldman, P. L.;Tetrahedron Lett. 1991, 32, 875-878). N.sup.α-(tert-butyloxycarbonyl)-L-ornithine tert-butyl ester (5.80 gm) wasdissolved in 100 ml chloroform and added to a solution of 5.70 gm ofcalcium carbonate and 2.2 ml of thiophosgene (28.7 mmol) dissolved in100 ml of water. The mixture was stirred vigorously overnight. The nextday the reaction mixture was filtered and the layers were allowed toseparate. The aqueous layer was extracted with chloroform (2×50 ml) andthe combined organic layers were dried (MgSO₄) and concentrated to anoil by evaporation at reduced pressure. The residue was taken up in drymethanol (200 ml) and cooled to 0° C. Ammonia gas was then passedthrough the solution for 20 minutes, and the solution was stirred for 3hours at 0° C. Following reaction with ammonia, the solvent wasevaporated under reduced pressure and the residue was dissolved in ethylacetate:hexane (4:1). That solution was chromatographed on a column ofsilica gel (25 cm×30 mm) using the same ethyl acetate/hexane mixture aseluent. Fractions of approximately 5 ml were collected, and thosecontaining product were identified by thin layer chromatography (seeabove, R_(f) =0.30). Product-containing fractions were pooled andevaporated to dryness under reduced pressure to yield N.sup.α-(tert-butyloxycarbonyl)-N.sup.δ -(thioureido)-L-norvaline tert-butylester in 70% yield. ¹³ C NMR (DCCl₃) 183.4-180.4 (one carbon),17.1-6,155-9, 82.2-80.0, 53.6-52.5 (one carbon), 44.6-43.1 (one carbon),30.4, 28.1, 27.8, 24.6.

N.sup.α -(tert-butyloxycarbonyl)-N.sup.═-(N-tert-butyloxycarbonyl-S-methyl) isothioureido-L-norvaline-tert-butylester

A solution of N.sup.α -(tert-butyloxycarbonyl)-N.sup.δ-(thioureido)-L-norvaline-tert-butylester (2.35 g, 6.62 mmol) andiodomethane (1 ml, 16.8 mmol) in CH₃ CN (15 ml) was stirred at 23° C.for 16 hours. The solution was concentrated and the residue dissolved indioxane (16 ml). To that solution was added saturated NaHCO₃ in water(16 ml) and di-tert-butyl pyrocarbonate (2.08 g., 9.54 mmol). Thereaction mixture was vigorously stirred at room temperature for 12hours. The dioxane was removed via evaporation under reduced pressure,and the aqueous solution was extracted with ethyl acetate (3×75 ml). Thecombined organic layers were dried over MgSO₄ and evaporated underreduced pressure. The residue was chromatographed on silica gel usinghexane/ethyl acetate (3:1) (R_(f) =0.50) to provide the product as anoil (3.08 g., 70%): ¹³ C NMR (DCCl₃) δ 173.0, 171.1, 161.8, 155.0, 81.7,79.3, 78.7, 53.0, 42.9, 29.7, 28.0, 27.9, 24.8, 13.2.

N.sup.δ -(S-methyl)-isothioureido-L-norvaline

The compound prepared in the previous step (0.470 g, 10.2 mmol) wasdissolved in 4N HCl/dioxane (3 ml) and stirred at room temperature for20 hours. The reaction mixture was diluted with ethyl ether (10 ml), andthe solvents were evaporated under reduced pressure. The addition andremoval of ethyl ether was repeated two more times. Methanol (10 ml) wasthen added and evaporated under reduced pressure to provide N.sup.δ-(S-methyl)-isothioureido-L-norvaline, also calledS-methyl-L-thiocitrulline, as a hydrochloride salt. (282 mg, 89.5%). ¹HNMR (D₂ O) δ 1.5-1.9(m,4H), 2.36(s,3H) , 3.23(t,2H) , 3.88(t,1H); massspectrum, 206(MH⁺).

Reference Example I

S-Methyl-D-thiocitrulline was prepared as described forS-methyl-L-thiocitrulline (Example I) except that the starting materialwas N.sup.δ -(benzyloxycarbonyl)-D-ornithine (Sigma Chemicals, Inc.).

EXAMPLE II Synthesis of S-Methyl-N.sup.ε -(thioureido)-L-norleucine(S-Methyl-L-homothiocitrulline)

N.sup.α -(Benzyloxycarbonyl)-L-lysine tert-butyl ester: N.sup.α-(Benzyl-oxycarbonyl)-L-lysine (5.0 gm, 17.84 mmol) was dissolved in 268ml of tert-butyl acetate containing 2.80 ml of perchloric acid (69-72%aqueous solution). The solution was stirred for 2 days at roomtemperature and then was extracted with water (3×200 ml). The combinedaqueous layers were adjusted to pH 10 with 50% sodium hydroxide. Theaqueous solution was then extracted with ethyl acetate (3×200 ml) andthe organic layers dried over MgSO₄. After filtration to remove MgSO₄,the solvent was removed by evaporation at reduced pressure to provideN.sup.α -(benzyloxycarbonyl)-L-lysine tert-butyl ester as a clear oil(2.16 gm, 36%). ¹ H NMR (CDCl₃) 1.35 (s,9H), 1.40-2.0 (m,6H), 2.60(t,2H), 4.10 (m, 1H), 4.98 (s,2H), 5.29 (bs,1H), 7.35 (s,5H).

N.sup.ε -(Thioureido)-N.sup.α -(benzyloxycarbonyl)-L-norleucinetert-butyl ester: N.sup.α -(Benzyoxycarbonyl)-L-lysine tert-butyl ester(2.16 gm, 6.43 mmol) was dissolved in 30 ml of chloroform and added to aprepared solution of calcium carbonate (1.80 g, 18.0 mmol) in 30 ml ofwater containing 0.70 ml of thiophosgene; the mixture was stirred for 6hours at room temperature. The reaction mixture was then filtered andthe layers separated. The aqueous layer was extracted with chloroform(2×30 ml) and the combined organic layers were dried over MgSO₄. Afterfiltration to remove MgSO₄, the dry solution was evaporated underreduced pressure to yield a clear oil. The residue was dissolved in 55ml of methanol and cooled to 0° C. Ammonia gas was passed into thesolution for 15 minutes and the reaction mixture was stirred for 3 hoursat 0° C. The methanol was then evaporated at reduced pressure and theresidue was chromatographed on a column of silica gel (25 cm×30 mm)using ethyl acetate:hexane (4:1) (R_(f) =0.33) as the eluent. Fractionscontaining product were identified by thin layer chromatography and werepooled. Evaporation of the product-containing fractions yielded N.sup.α-(benzyloxycarbonyl)-N.sup.ε -thioureido-L-norleucine tert-butyl esteras a foamy solid. ¹³ CNMR (CDCl₁₃) δ 182.70-179.6 (one carbon), 171.35,155.99, 135.77, 129.19, 128,85, 127.08, 126.61, 81.92, 68.5-64.65 (onecarbon) 54.73-52.97 (one carbon), 46.30-41.50 (one carbon), 30.10,28.40, 26.73, 25.0.

N.sup.ε -S-Methyl)-thioureido-L-norleucine(S-Methyl-L-homothiocitrulline): N.sup.α -(Benzyloxycarbonyl)-N.sup.ε-thioureido-L-norleucine tert-butyl ester (340 mg, 0.86 mmol) wasstirred with iodomethane (0.1 ml) in acetonitrile (2 ml) for 16 hours atroom temperature. The solvent was then evaporated under reducedpressure, and the residue was treated with 4M HCl/dioxane (5 ml) at 75°C. After 1 hour the solvent was evaporated and the resulting residue wastriturated with ethyl ether (10 ml). The ether was evaporated andmethanol (10 ml) was added and evaporated to yield a hygroscopic solid(226 rag, 90%); ¹ H NMR (D₂ O) δ 1.34-1.80 (m, 6H), 2.42 (S,3H), 3.20(m,2H), 3.86 (t, 1H); mass spectrum, m/e 220 (M+H⁺).

EXAMPLE III Inhibition of Smooth Muscle Nitric Oxide Synthase byS-Methyl-L-thiocitrulline and Other Compounds

The activity of S-methyl-L-thiocitrulline, L-thiocitrulline, andNG-methyl-L-arginine as inhibitors of smooth muscle nitric oxidesynthase was determined in vitro by monitoring the conversion of [¹⁴C]arginine to [¹⁴ C]citrulline.

Smooth muscle nitric oxide synthase, an example of induced nitric oxidesynthase (iNOS) was obtained as follows: aortic smooth muscle cells werecultured by explanting segments of the medial layer of the aortae ofadult male Fischer 344 rats. Aortae were removed aseptically and freedof adventitial and endothelial cells by scraping both the luminal andabluminal surfaces. Medial fragments (1-2 mm) were allowed to attach todry Primaria 25 cm² tissue culture flasks (Falcon; Oxnard, Calif.) whichwere kept moist with growth medium until cells emerged. Cultures werefed twice weekly with medium 199 containing 10% fetal bovine serum, 25mM HEPES, 2 mM L-glutamine, 40 μg/ml endothelial cell growth supplement(Biomedical Technologies; Stoughton, Mass.) and 10 μg/ml gentamyocin(GIBCO; Grand Island, N.Y.). When primary cultures became confluent,they were passaged by trypsinization. Cells in passage 10-15 were seededat 20,000/well. When the cells became confluent (density of 60-80×10³cells in a well), the medium was removed by suction and fresh mediumconsisting of 200 μl of RPMI 1640 (Whittaker Laboratories) containing10% bovine calf serum, 25 mM HEPES buffer (pH 7.4), 2 mM glutamine, 80U/ml penicillin, 80 μM/ml streptomycin, 2 μg/ml fungizone, 40 ng/mlinterleukin-1 and 50 ng/ml interferon-gamma was introduced.Interleukin-1 and interferon-gamma are effective inducers of iNOS.

In a final volume of 200 μl, the reaction mixtures contained thefollowing: 20 mM sodium HEPES, pH 7.15, 0.1 mM EGTA (ethylene glycolbis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), 0.1 mMdithiothreitol, 100 μM tetrahydrobiopterin, 500 μM NADPH, 25 μM FAD, 25μM FMN and 0.03-0.05 units of nitric oxide synthase (1 unit equals theamount of enzyme necessary to convert 1 nmol of arginine to citrullineand nitric oxide per min). The reaction was begun by adding to thereaction mixture 20 μM L-[¹⁴ C]arginine and, except in the case of thecontrol, 0.1 μM (S-methyl-L-thiocitrulline, 1.0 μMS-methyl-L-thiocitrulline or 10 μM S-methyl-L-thiocitrulline. Thereaction mixtures were maintained at 25° C. and at 3.5, 7.0, and 10.5minutes 50 μl portions were removed from each reaction mixture and addedto 200 μl of 100 mM sodium HEPES, pH 5.5 containing 5 mM EGTA. Thedecrease in pH stops the reaction. The reaction mixtures were thenplaced in a boiling water bath for 1 minute which caused the protein toprecipitate; the precipitate was removed by centrifugation. A 225 μlportion of the supernatant was then removed and applied to a smallcolumn of Dowex 50 (Na+ form, 200-400 mesh, 0.5×3.5 mm). L-[¹⁴C]citrulline, a product of the reaction, was eluted from the columnsusing 2.0 ml of water. The eluant was collected directly into ascintillation vial, 10 ml of scintillation fluid (Econo-Safe, ResearchProducts International Corp.) were added and the contained radioactivitywas determined by liquid scintillation counting. Knowing the specificactivity of the L-arginine in the original reaction mixture, it waspossible to convert the cpm citrulline data to pmol product.

Results are shown in FIG. 1 wherein product formation was plotted as afunction of time. In FIG. 1 the line denoted by open circles is thearginine control, the line denoted by filled in circles is for theexperiment with 0.1 μM S-methyl-L-thiocitrulline, the line denoted byopen triangles is for the experiment with 1.0 μMS-methyl-L-thiocitrulline, and the line denoted by filled in trianglesis for the experiment with 10 μM S-methyl-L-thiocitrulline.

As shown in FIG. 1, in the absence of test compound (20 μM argininecontrol line) product formation was constant over a 10.5 minute periodobserved. Addition of 0.1, 1.0 and 10 μM S-methyl-L-thiocitrullinecaused about 10%, 32% and 84% inhibition, respectively.

Under similar reaction conditions, 10 and 30 μM L-thiocitrulline caused34 and 70% inhibition respectively, showing that L-thiocitrulline isless potent than S-methyl-L-thiocitrulline.

Addition of 100 μM NG-methyl-L-arginine (L-E, the prototypic nitricoxide synthase inhibitor) caused inhibition approximately equal to thatof 10 μM S-methyl-L-thiocitrulline.

EXAMPLE IV Inhibition of Smooth Muscle Nitric Oxide Synthase byS-Methyl-L-homothiocitrulline and L-Homothiocitrulline

Using the same protocol as described for the studies described inExample I, 100 μM homothiocitrulline and 100 μMS-methyl-L-homothiocitrulline were compared as inhibitors of smoothmuscle nitric oxide synthase. Time points were taken at 4, 8 and 12minutes rather than at 3.5, 7 and 10.5 minutes. The results of the studywith L-homothiocitrulline and S-methyl-L-homothiocitrulline are shown inFIG. 2. In FIG. 2, the line denoted by the open circles is the controlindicating the rate of citrulline formation in the absence ofinhibitors. The line denoted by the filled circles is for the experimentwith 100 μM L-homothiocitrulline, and the line denoted by the opentriangles is for the experiment with 100 μMS-methyl-L-homothiocitrulline. The experiment shows that whereasL-homothiocitrulline causes significant inhibition under thesecircumstances, an identical concentration ofS-methyl-L-homothiocitrulline causes complete inhibition; theS-methyl-L-homothiocitrulline is therefore a more potent inhibitor thanL-homothiocitrulline.

EXAMPLE V The Effect of S-Methyl-L-thiocitrulline on Blood Pressure

To test the ability of S-methyl-L-thiocitrulline to block basal nitricoxide formation in vivo, its effects were tested in anesthetizedSprague-Dawley rats. Rats (250-300 gm) were anesthetized with Inactin(100 mg/kg i.p.) and placed on a heated surgical table to maintain bodytemperature of 36.5° C. Femoral arterial and venous catheters wereimplanted (tips were distal to the renal artery) for measurement ofblood pressure and infusion of compounds, respectively. Blood pressurewas measured using a pressure transducer (Cobe, Inc.) connected to amicrocomputer using data acquisition software (AT-CODAS; Data QInstruments, Akron, Ohio). After waiting several minutes to establish astable baseline blood pressure, S-methyl-L-thiocitrulline (20 mg/kg) wasgiven by bolus injection through the venous catheter. The solutionadministered was 100 mM. Changes in systolic, diastolic, and meanarterial pressure were monitored for approximately 25 minutes. Theeffects on systolic blood pressure are shown in FIG. 3. As shown,S-methyl-L-thiocitrulline caused a significant (70 mm Hg, 50%) increasein blood pressure which lasted 10-15 minutes. Under similarcircumstances, NG-methyl-L-arginine, the prototype inhibitor caused asomewhat smaller increase in blood pressure, which lasted about the sametime. There was no significant effect on heart rate. The studiesindicate that S-methyl-L-thiocitrulline is an effective pressor agent invivo. Based on earlier studies with NG-methyl-L-arginine, the prototypicnitric oxide synthase inhibitor, S-methyl-L-thiocitrulline is viewed asblocking the basal release of nitric oxide from vascular endothelialcells. Decreased release of basal nitric oxide removes an importantvasodilatory mediator involved in normal blood pressure regulation andresults in increased vascular tone. Increased vascular tone(vasoconstriction) increases systemic vascular resistance; bloodpressure consequently increases.

EXAMPLE VI The Effect of S-Methyl-L-thiocitrulline on Blood Pressure inCytokine Therapy

A human is continuously administered interleukin-2 (1×10⁶ units) for 5days. S-Methyl-L-thiocitrulline (2 to 20 mg/kg/day) is administered bycontinuous infusion at a rate sufficient to maintain a systolic bloodpressure of 80-120 mm Hg. The severe hypotension characteristic of theend of interleukin-2 therapy is significantly reduced.

EXAMPLE VII The Effect of S-Methyl-L-thiocitrulline on IncreasingResponse to Pressor Agents

Sprague-Dawley rats are injected intraperitoneally with bacteriallipopolysaccharide (a bacterial endotoxin) (15 mg/kg) alone, togetherwith phenylephrine (6 μg/kg), and together withS-methyl-L-thiocitrulline (20 mg/kg) and phenylephrine (6 μg/kg). Thetreatment with the combination of S-methyl-L-thiocitrulline andphenylephrine significantly reduces the fall in blood pressure frombacterial lipopolysaccharide administration to a greater degree thanphenylephrine alone.

EXAMPLE VIII Inhibition of Brain Nitric Oxide Synthase byS-Methyl-L-thiocitrulline and Other Compounds

The activity of S-methyl-L-thiocitrulline, L-thiocitrulline, andNG-methyl-L-arginine as inhibitors of rat brain nitric oxide synthasewas determined in vitro by monitoring the conversion of [¹⁴ C]arginineto [¹⁴ C]citrulline.

Purified rat brain nitric oxide synthase, isolated as described(McMillan, K., Bredt, D. S., Hirsch, D.J., Synder, S. H., Clark, J. E.,and Masters, B. S., Proc. Natl. Acad. Sci. U.S.A. Vol 89, pp.,11141-11145, December 1992) was obtained from Dr. Bettie Sue Masters,The University of Texas health Science Center at San Antonio, Departmentof Biochemistry, 7703 Floyd Curl Drive, San Antonio, Tex. 78284.

In a final volume of 200 μl, the reaction mixtures contained thefollowing: 20 mM sodium HEPES, ph 7.5, 0.1 mM EGTA, 0.1 mMdithiothreitol, 10 μg/ml calmodulin, 2 mM calcium chloride, 100 μMtetrahydrobiopterin, 100 μg/ml bovine serum albumin, 500 μM NADPH, 25 μMFAD, 25 μM FMN and 0.03-0.05 units of nitric oxide synthase (1 unitequals the amount of enzyme necessary to convert 1 nmol of arginine tocitrulline and nitric oxide per rain). The reaction was begun by addingto the reaction mixture 20 μM L-[¹⁴ C]arginine and, except in the caseof the control, 0.1 μM S-methyl-L-thiocitrulline, 1.0 μMS-methyl-L-thiocitrulline or 10 μM S-methyl-L-thiocitrulline. Thereaction mixtures were maintained at 25° C. and at 3.5, 7.0, and 10.5minutes 50 μl portions were removed and added to 200 μl of 100 mM sodiumHEPES, pH 5.5, containing 5 mM EGTA. The decrease in pH and the bindingof calcium by EGTA stops the reaction. The reaction mixtures were thenplaced in a boiling water bath for 1 minute which caused the protein toprecipitate; the precipitate was removed by centrifugation. A 225 μlportion of the supernatant was then removed and applied to a smallcolumn of Dowex 50 (Na+ form, 200-400 mesh, 0.5×3.5 mm). L-[¹⁴C]Citrulline, a product of the reaction, was eluted from the columnsusing 2.0 ml of water. The eluant was collected directly into ascintillation vial, 10 ml of scintillation fluid (EconoSafe, RPI Corp,Mt. Prospect, Ill.) were added and the contained radioactivity wasdetermined by liquid scintillation counting. Knowing the specificactivity of the L-[¹⁴ C]arginine in the original reaction mixture, itwas possible to convert the cpm citrulline data to pmol product. Resultsare shown in FIG. 4 wherein product formation is plotted as a functionof time. As shown in FIG. 4, the line denoted by open circles is thecontrol, the line denoted by filled in circles is for the experimentwith 0.1 μM S-methyl-L-thiocitrulline, the line denoted by open intriangles is for the experiment with 1.0 μM S-methyl-L-thiocitrulline,and the line denoted by filled in triangles is for the experiment with10 μM S-methyl-L-thiocitrulline.

As shown in FIG. 4, in the absence of inhibitor (20 μM arginine controlline), product formation is constant over the 10.5 minute periodobserved. Addition of 0.1, 1.0 and 10 μM S-methyl-L-thiocitrullinecaused about 5%, 65% and 95% inhibition respectively.

Under identical reaction condition, L-thiocitrulline at 10 and 30 μMcaused 50 and 75% inhibition, respectively, showing thatS-methyl-L-thiocitrulline is a more potent inhibitor than isL-thiocitrulline.

Addition of 100 μM NG-methyl-L-arginine (L-NMA, the prototypic nitricoxide synthase inhibitor) also caused substantial inhibition, but L-NMAwas much less effective than 100 μM S-methyl-L-thiocitrulline (notshown).

EXAMPLE IX Inhibition of Brain Nitric Oxide Synthase byS-Methyl-L-homothiocitrulline and L-Homothiocitrulline

Using the same protocol as for the studies described in Example VIII,L-homothiocitrulline and S-methyl-L-homothiocitrulline were compared asinhibitors of brain nitric oxide synthase. Time points were taken at 4,8 and 12 minutes rather than at 3.5, 7 and 10.5 minutes. The results ofthe study with L-homothiocitrulline and S-methyl-L-homothiocitrullineare shown in FIG. 5. In FIG. 5, the line denoted by the open trianglesis the control indicating the rate of citrulline formation in theabsence of inhibitors. The line denoted by the open circles is for theexperiment with 100 μM L-homothiocitrulline, and the line denoted by thefilled in circles is for the experiment with 100 μMS-methyl-L-homothiocitrulline. The experiment shows that whereasL-homothiocitrulline causes significant inhibition under thesecircumstances, an identical concentration ofS-methyl-L-homothiocitrulline causes complete inhibition;S-methyl-L-homothiocitrulline is therefore a more potent inhibitor thanL-homothiocitrulline.

EXAMPLE X Treatment of Stroke

Both common carotid arteries are ligated in two groups of femaleSprague-Dawley CFY rats. After 10 minutes, ligations are released andflow is again allowed.

In the case of one group, S-methyl-L-thiocitrulline (20 mg/kg) isadministered by bolus injection through a venous catheter 30 minutesafter occlusion of the arteries. In the case of the other group, notherapeutic agent is administered.

Histological analysis of stroke volume 24 hours following arteryocclusion shows significant reduction in stroke volume for the groupadministered S-methyl-L-thiocitrulline compared to the group receivingno treatment.

EXAMPLE XI Treatment of Inflammation

Sprague-Dawley rats are injected with 0.25 cc air subdermally in thedorsal area in accordance with an air pouch inflammatory model (Selye,H., Proc. Soc. Exper. Biol. and Med., 82, 328-333 (1953)). Into the airpouch formed, an inflammatory stimulus, croton oil (0.5% in 0.5 ml cornoil), is injected. Simultaneously, the rats in one group areadministered intraperitoneally S-methyl-L-thiocitrulline (20 mg/kg) andthis administration is repeated every 12 hours for 5 days. The rats inanother group are not administered S-methyl-L-thiocitrulline. At the endof the 5 days, the group of rats given S-methyl-L-thiocitrulline havesignificantly less inflammation than the rats in the other group.

When the same amount of S-methyl-L-homothiocitrulline is substituted forL-thiocitrulline in Examples V, VI, VII, X and XI, similar results areobtained.

Many variations of the above will be obvious to those skilled in theart. Thus, the invention is defined by the claims.

What is claimed is:
 1. A method for treatment of a subject for systemic hypotension caused by pathological overproduction of nitric oxide from arginine by nitric oxide synthase induced in said subject with a cytokine or by a bacterial endotoxin, said method comprising administering to a subject in need of said treatment a conventional therapeutically effective amount of at least one α₁ -adrenergic agonist and an amount of a compound effective to restore vascular contractile sensitivity to the effects of said α₁ -adrenergic agonist, said compound which is effective to restore vascular contractile sensitivity to the effects of said α₁ -adrenergic agonist being a physiologically active compound including an N.sup.δ -substituted ornithine moiety or an N.sup.ε -substituted lysine moiety or a monoalkyl carbon-substituted N.sup.δ -substituted ornithine moiety or an N.sup.ε -substituted lysine moiety, having the formula ##STR3## wherein R is (CH₂)_(y) CH₃ or H, R' is CH₂ or C(H)(CH₂)_(y) CH₃, and R" is CH₂ or C(H)(CH₂)_(y) CH₃, with y ranging from 0 to 5, and x is 0 or 1 and wherein none or only one of R, R' and R" provides an alkyl substituent on ornithine or lysine moiety, and wherein Q is alkyl having 1 to 5 carbon atoms, and physiologically acceptable acid addition salts thereof.
 2. The method of claim 1 wherein the compound is S-methyl-L-thiocitrulline or a physiologically acceptable acid addition salt thereof.
 3. The method of claim 1 wherein the compound is S-methyl-L-homothiocitrulline or a physiologically acceptable acid addition salt thereof. 